The Effect of Mechanics on Migration, Morphology and Matrix Production by Primary Human Corneal Fibroblasts: Long-Term Dynamic Observation

Doctor of Philosophy Mechanical load is believed to play a critical role in tissue formation, growth, maintenance, and disease in vertebrate animals. Although many studies have investigated the effect of mechanical stimuli on biological processes, the dynamics of these processes have not been closely explored. Live long-term observation of the collective behavior of Primary Human Corneal Fibroblasts (PHCFs) exposed to mechanical load could provide important insight into the mechanisms used by fibroblasts while they migrate, produce multilayered constructs and manage collagen deposition during the synthesis of organized ExtraCellular Matrix (ECM). Previous studies have shown that PHCFs produce locally organized and aligned ECM similar to developing corneas when cultured in the presence of stabilized ascorbic acid. In this study, DIC imaging, live cell fluorescent labeling, traction force microscopy and our previously designed mechanobioreactor were employed to locally and globally capture PHCF's behavior under cell culture conditions for up to two weeks. We have directly tracked and observed the morphology and migration of PHCFs under physiological conditions in the presence of uniaxial mechanical load directly on the microscope. The behavior of human corneal fibroblasts is observed from initial seeding through matrix production. The orientation of cells, the magnitude and direction of their velocity as well as forces exerted by PHCFs were tracked on substrates every 6 minutes. The analyzed data demonstrates that corneal fibroblasts are induced to align on loaded substrates at a relatively fixed angle to the applied force while corneal fibroblasts on unloaded substrates exhibit only local alignment. We conclude that applied external mechanical loads significantly affect the patterns of cell migration and culture morphology and that load abrogates local guidance signals. Tissue engineers should be able to take advantage of such mechanical guidance to produce highly organized tissues such as the corneal stromal matrix.